The mRNA expression profiles of GSTCD, HHIP, THSD4, TNS1, HTR4, AGER and NOTCH4 were determined in human lung tissue and primary cell samples using RT-PCR, including RNA from lung (Ambion/ABI), brain, airway smooth muscle cells41 and human bronchial epithelial cells (Clonetics42). Peripheral blood mononuclear cells were isolated from whole blood using 6% (w/v) dextran and 42%–51% (v/v) Percoll gradients (Sigma). Ethical approval for the use of primary cells was obtained from the local ethics committees. Total RNA was extracted from samples using an RNeasy kit (Qiagen) as directed by the manufacturer. cDNA was generated from 1 μg of RNA template using random hexamers and a SuperScript kit (Invitrogen) as directed by the manufacturer. PCR assays were designed to cross intron-exon boundaries and where splice variation was known, in order to detect all variants. Primer sequences are given in Supplementary Table 6. All PCR was done using Platinum Taq High Fidelity (Invitrogen) with 100 ng of cDNA template in a 25-μl reaction. Cycling conditions were as follows: 94 °C for 3 min, 35 cycles of 94 °C for 45 s, 55 °C for 30 s, and 72 °C for 90 s.
